Metabolic profiling as a means of characterizing plant-associated microbial communities

Abstract Microbial communities from leaf and root habitats associated with sugar beet ( Beta vulgaris ) were characterized according to their capacity to metabolize a range of 95 sole carbon sources available in a commercial assay, GN-BIOLOG MicroPlates. Metabolic profiling was assessed as a method for evaluating perturbation of microbial communities of glasshouse-grown sugar beet inoculated with a genetically modified microorganism. This technique has allowed microbial communities, colonising the immature leaves of treated and untreated plants to be differentiated, although no differences were observed when plants inoculated with genetically modified microorganisms and unmodified inoculated plants were compared. As plants developed and differentiated, the carbon utilization patterns observed allowed communities to be grouped according to the habitat from which they were isolated, irrespective of treatment. These studies demonstrate that the genetically modified microorganism, introduced as a seed dressing colonised developing immature tissue throughout the 231-day study but did not disrupt the natural succession of microbial communities in glasshouse-grown sugar beet plants.     

Runoff losses of Pseudomonas aureofaciens (lacZY) from soil

Abstract: Leaching of genetically modified microorganisms (GMMs) through soil profiles is generally not a significant concern, since GMMs typically remain near the soil surface following application. The presence of high numbers of GMMs at the soil surface, however, suggests that losses via runoff may occur. Traditional methods of plating nonlabeled bacteria lack precision and are thus seldom used to monitor runoff losses. To examine whether lac ZY, a common genetic marker, could be used to evaluate bacterial runoff from soil, a lac ZY⁺ strain of Pseudomonas aureofaciens 3732 RN-L11 was used at three different pH levels, with and without wheat ( Triticum aestivum L.) cover in a greenhouse experiment. Twelve times over a 245 day period, soils were subjected to simulated rainfall of 84 mm h⁻¹ for a 15 min duration. Runoff losses and survival were quantified at each time. Pseudomonas aureofaciens survived for the longest period at a soil pH of 7; survival was reduced at lower pHs. The number of cells in runoff were usually related to the number of cells surviving in the soil. When high soil populations were present, runoff losses often exceeded 10¹⁰ cfu event⁻¹. When the soil population declined to low or undetectable levels, the runoff contained fewer organisms. Runoff losses of 10⁸ cfu event⁻¹, however, were observed during one runoff event even when the soil population was below the detection limit. This study indicates that lac ZY is an effective marker, and that runoff of GMMs may be an important mechanism for movement to nontarget environments.     

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions

Background

According to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called ‘minimum required performance limit’ (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.

Results

We developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section ‘Availability of supporting data’ for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.

Conclusions

The aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in —but not limited to— the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0407-x) contains supplementary material, which is available to authorized users.     

Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the field

Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25:tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25:tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25:tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions.     

The long and winding road from the research laboratory to industrial applications of lactic acid bacteria

Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology.     

Ecological risk assessment of genetically modified crops based on cellular automata modeling

The assessment of ecological risk in genetically modified (GM) biological systems is critically important for decision-making and public acceptance. Cellular automata (CA) provide a potential modeling and simulation framework for representing relationships and interspecies interactions both temporally and spatially. In this paper, a simple subsystem contains only four species: crop, target pest, non-target pest and enemy insect, and a three layer arrangement of L × L stochastic cellular automata with a periodic boundary were established. The simulation of this simplified system showed abundant and sufficient complexity in population assembly and densities, suggesting a prospective application in ecological risk assessment of GM crops.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

厌氧氨氧化菌的代谢途径及其关键酶的研究进展

作为新型生物脱氮工艺的代表,厌氧氨氧化反应的代谢功能菌——厌氧氨氧化(Anammox)菌也逐渐成为研究的热点。本文介绍了10种Anammox菌的发现过程,阐述了Anammox菌的关键酶的研究进展。通过对Candidatus"Brocadia anammoxidans"和Candidatus"Kuenenia stuttgartiensis"菌的代谢途径的分析和推理,综述了化学计量模型、生化反应模型和能量代谢模型,并提出了一种可能存在的基于关键酶的厌氧氨氧化菌微界面代谢新途径。     

Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community level

Abstract: Substrate utilization of microbial cells extracted from soil with a 0.85% aqueous sodium chloride solution, was determined to estimate effects on soil microorganisms at the community level with microtiter plates (Biolog GN®) containing 95 different sources of organic carbon. A consistent pattern of utilized substrates was obtained after 24 h of microtiter plate incubation at 28°C. The absorbance values (OD₅₉₀) obtained from a microtiter plate reader after background correction were transformed by using the average absorbance values of oxidized substrates as a threshold to distinguish between well utilized and poorly or non-utilized substrates and thereby reduce variances between replicates. Doubling times of the extracted soil microorganisms in the microtiter plates were tested with 12 substrates and ranged from 1.96 h to 3.23 h, depending on the carbon source. The carbon source utilization assay was used to assess the effects of soil inoculation with Corynebacterium glutamicum with and without a genetically engineered plasmid (pUN1; 6.3 kb), which encoded for the synthesis of the mammalian protease inhibiting peptide, aprotinin. Additionally, aprotinin itself was added at two concentrations to soil samples. An identical decrease in the number of carbon sources utilized, especially carbohydrates, occurred upon soil inoculation with both C. glutamicum strains after inoculation with 10⁶ cells g⁻¹ soil. This effect was only detectable during the first three weeks of incubation, as long as cell numbers of C. glutamicum (pUN1) were above 10⁵ cfu g⁻¹. Soil amendment with aprotinin resulted in utilization of additional substrates, most of them carbohydrates. With 0.1 mg aprotinin g⁻¹ soil this stimulation lasted 2 days and with 10 mg g⁻¹ it lasted for 7 days.     

A copolymer analysis approach to estimate the neutral sugar distribution of sugar beet pectin using size exclusion chromatography

Partially degraded sugar beet (Beta vulgaris) pectins were characterised in terms of galacturonic acid, neutral sugar and ferulic acids contents. It was shown that the total neutral sugar content is correlated with the ferulic acid content. One pectin (C) was further characterised by size exclusion chromatography coupled to refractive index and UV detectors (SEC-RI-UV). This gave the opportunity to estimate how the ferulic acid and neutral sugar contents changed with hydrodynamic radius. Pectin C was found to be heterogeneous in composition with neutral sugar-rich fractions of both high and low hydrodynamic radii. A neutral sugar-poor fraction was found at intermediate hydrodynamic radii.     

A review of food-grade vectors in lactic acid bacteria: from the laboratory to their application

Lactic acid bacteria (LAB) have a long history of use in fermented foods and as probiotics. Genetic manipulation of these microorganisms has great potential for new applications in food safety, as well as in the development of improved food products and in health. While genetic engineering of LAB could have a major positive impact on the food and pharmaceutical industries, progress could be prevented by legal issues related to the controversy surrounding this technology. The safe use of genetically modified LAB requires the development of food-grade cloning systems containing only the DNA from homologous hosts or generally considered as safe organisms, and not dependent antibiotic markers. The rationale for the development of cloning vectors derived from cryptic LAB plasmids is the need for new genetic engineering tools, therefore a vision from cryptic plasmids to applications in food-grade vectors for LAB plasmids is shown in this review. Replicative and integrative vectors for the construction of food-grade vectors, and the relationship between resistance mechanism and expression systems, will be treated in depth in this paper. Finally, we will discuss the limited use of these vectors, and the problems arising from their use.     

Synthesis of two trisaccharides related to the triterpenoid saponins isolated from Solanum lycocarpum

Chemical synthesis of two trisaccharides related to the triterpenoid saponins isolated from Solanum lycocarpum from commercially available d-Glc, d-Gal and l-Rha have been achieved by following concise and high-yielding route. The target trisaccharide 1 has been made by following a bis-glycosylation approach that has minimized the protecting group manipulations up to great extent. The trisaccharides have been synthesized in the form of their p-methoxyphenyl (OMP) glycosides to leave the scope for further glycoconjugates formation by the selective removal of the OMP glycoside and trichloroacetimidate chemistry. La(OTf)₃ has been used successfully as the promoter for the NIS mediated activation of thioglycosides.     

用于微生物多样性分析的棉田土壤总DNA的提取

目的：探讨适用于微生物多样性研究的棉田土壤微生物总DNA提取方法。方法：采用4种方法提取不同连作和轮作处理的棉田土壤微生物总DNA,比较其纯度、产率、片段大小,并应用ARDRA技术验证其质量。结果：其中3种方法均可获得23kb的DNA片段,但不同方法提取的DNA的产率和纯度上有明显差异。改良CTAB-SDS法提取的DNA完整性好,得率为24.20μg.g-1干土,纯化后A260/A280和A260/A230为分别为1.80和1.70,纯化回收率可达70.1%,完全适用于后续的PCR分析。结论：采用该法提取棉田土壤总DNA简便而高效。对该法提取获得的棉田土壤微生物总DNA进行ARDRA和DGGE分析,所得图谱能较全面地反映不同处理间微生物多样性及群落结构的差别,为不同栽培体系下棉田土壤微生物的分子生态学研究提供了基础。     

Susceptibility of cell substrates to PrPSc infection and safety control measures related to biological and biotherapeutical products

Concerns over the potential for infectious prion proteins to contaminate human biologics and biotherapeutics have been raised from time to time. Transmission of the pathogenic form of prion protein (PrPSc) through veterinary vaccines has been observed, yet no human case through the use of vaccine products has been reported. However, iatrogenic transmissions of PrPSc in humans through blood components, tissues, and growth hormone have been reported. These findings underscore the importance of reliable detection or diagnostic methods to prevent the transmission of prion diseases, given that the number of asymptomatic infected individuals remains unknown, the perceived incubation time for human prion diseases could be decades, and no cure of the diseases has been found yet. A variety of biochemical and molecular methods can selectively concentrate PrPSc to facilitate its detection in tissues and cells. Furthermore, some methods routinely used in the manufacturing process of biological products have been found to be effective in reducing PrPSc from the products. Questions remain unanswered as to the validation criteria of these methods, the minimal infectious dose of the PrPSc required to cause infection and the susceptibility of cells used in gene therapy or the manufacturing process of biological products to PrPSc infections. Here, we discuss some of these challenging issues.     

A bioprocessing strategy that allows for the selection of Cr(VI)-reducing bacteria from soils

Anaerobic bacteria that reduce hexavalent chromium [Cr(VI)] to trivalent [Cr(III)] are common in soils and were used to develop a bioprocess employing a selection strategy. Indigenous Cr(VI)-reducers were enriched from Cr(VI)-contaminated soil under anaerobic conditions. The mixed culture was then tested for Cr(VI)-reducing activity in a chemostat, followed by transfer to a 1-L packed-bed bioreactor operated at 30°C for additional study. The support material used in the reactor consisted of 6-mm porcelain saddles. Cr(VI) concentrations in the liquid ranged from 140–750 mg L⁻¹. Cr(VI)-reducing bacteria were the dominant population with Cr(VI)-reduction rates of approximately 0.71 mg g⁻¹ dry cells h⁻¹ achieved at Cr(VI) concentrations of 750 mg L⁻¹. These results indicate a potential for selecting and maintaining indigenous Cr(VI)-reducers in a bioreactor for Cr(VI)-remediation of groundwater or soil wash effluents. Received 09 January 1996/ Accepted in revised form 15 November 1996     

A new method to determine DNA sugar conformation from the joint use of 2D and 3D NMR data

The use of sugar restraints has been proven essential for assessing DNAstructures through molecular modeling studies. We present a new methodcombining 2D (COSY and NOESY) and 3D (NOESY-NOESY) experiments, whereconstraints on either the phase angles or the difference between phase anglesof two residues are obtained from comparison of 2D NOE H1-H4intensities and 3D NOE intensities containing the H1-H4transfer. All experiments lead to restraints that match, proving the validityof the method.